Biodiversity, Bioprospecting and
Development
Kumar et al., J Biodivers Biopros Dev 2014, 1:3
http://dx.doi.org/10.4172/ijbbd.1000127
Research Article
Open Access
Evaluating the Diversity of Culturable Thermotolerant Bacteria from Four Hot
Springs of India
Murugan Kumar, Ajar Nath Yadav, Rameshwar Tiwari, Radha Prasanna and Anil Kumar Saxena*
Division of Microbiology, Indian Agricultural Research Institute (IARI), New Delhi 110012, India
*Corresponding
author: Anil Kumar Saxena, Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India, Tel: 91 11 25847649; Fax: 91 11
25846420; E-mail: [email protected]
Received date: Jul 04, 2014, Accepted date: Sep 27, 2014, Publication date: Sep 29, 2014
Copyright: © 2014 Kumar M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
The culturable bacterial diversity of four hot springs of India was analysed, employing different media and
screened for temperature tolerance (40°C - 70°C). Sixty morphotypes from Bakreshwar, 48 from Balrampur, 46 from
Chumathang and 52 from Vashist were obtained. A set of 112 isolates, tolerant to 45°C and above, were analysed
employing Amplified ribosomal DNA restriction analysis (ARDRA). Sequencing of 16S rRNA gene of the
representative isolates revealed that 86%, 93%, 44% and 44% of the isolates respectively from Bakreshwar,
Balrampur, Chumathang and Vashist, belonged to Firmicutes. Members of Actinobacteria were present in all the
four hot springs, while Proteobacteria were present only in Chumathang and Vashist and Bacteroidetes found only
in Bakreshwar. This is the first report of Aurantimonas and Brevundimonas in hot springs. Biolog analyses of three
isolates, growing at or above 60°C revealed unique abilities, in terms of utilization of substrates and resistance
patterns.
Keywords: ARDRA; Biolog; Culturable bacteria; 16S rRNA; Hot
springs; Thermotolerance
Abbreviations
ARDRA: Amplified ribosomal DNA restriction analysis; rRNA:
Ribosomal ribonucleic acid; PCR: Polymerase chain reaction; HGB:
Himalayan geothermal belt
Introduction
Among the many extreme environments, thermal springs are of
considerable interest to researchers worldwide. Microbial
communities present in such habitats constitute valuable sources for
various biotechnological products [1,2]. India is the home to several
hot springs, which have been less explored in terms of biotic
components [3], as there are very limited published literatures
available on the microbial diversity and their utility [3,4].
Worldwide, studies on microbial communities in hot springs have
mainly concentrated on habitats at low elevations like Yellowstone
National Park [5], Kamchatka in Russia, Iceland [6], Indonesia [7] and
Tunisia [2]. But, very little is known about the bacterial diversity in
thermal springs from highly elevated regions. The Himalayan
Geothermal Belts (HGB) contains nearly 150 thermal springs.
Chumathang in Jammu and Kashmir and Vashist in Himachal
Pradesh [8] representing high altitude are well known hot springs of
HGB. Bakreshwar is a well known hot spring representing plain
located in West Bengal which shows temperature in the range of 35°C
to 66.5°C [9]. Balrampur, located in Chattisgarh is the home to a low
altitude hot spring, which is less investigated. In the present
investigation, an attempt was made to compare the diversity of
culturable bacteria in these four hot springs and identify niche-specific
bacterial genera/groups.
J Biodivers Biopros Dev
ISSN: IJBBD, an open access journal
In the recent years there has been an increasing emphasis on
metagenomic approaches to analyse diversity [2,10,11]. Traditional
methods of isolation and culturing have several limitations and can be
considered inefficient in a way that they cannot recover symbiotic,
slow growing organisms and viable but non-culturable fractions that
are believed to make up the bulk of environmental samples [12].
Despite limitations, the advantage of culture based diversity studies
outweighs metagenomic approaches in the opportunity it offers to
generate a large and valuable germplasm. Also, the main advantage in
working with isolated strains is that they can be preserved for further
studies and can be explored for biotechnological applications, when
the need arises [13,14].
Advances in molecular biology techniques such as sequencing a
stable part of genetic code have provided an excellent opportunity for
complementing the identification and characterization of bacteria at
species and subspecies levels [15]. Candidate genes used for this study
included as many as 20 including 5S rRNA, 16S rRNA, 23S rRNA and
16-23S rRNA internal transcribed spacer (ITS) region. Among all
these genes, 16S rRNA is considered the best evolutionary
chronometer because (i) it is universally present in all bacteria; (ii) its
function over time has not changed, suggesting that random sequence
changes are a more accurate measure of time (i.e. evolution); and (iii)
the 16S rRNA gene (1,500 bp) is large enough for bioinformatic
analyses [16]. Amplified ribosomal DNA restriction analysis (ARDRA)
represents a further improvement in the analysis of 16S rRNA genes
and involves amplification of 16S rRNA gene followed by digestion of
amplified product using selected restriction enzymes and generation of
restriction patterns. Restriction patterns can then be compared [17]
and analysed using bioinformatic tools. The utility of ARDRA in
genotypic characterization of bacteria [18] and screening clone
libraries [19] for grouping them into phylogenetic clusters is
undisputed. ARDRA has been utilized in the past for identification of
bacteria up to species level also [20].
Volume 1 • Issue 3 • 1000127
Citation:
Kumar M, Nath Yadav A, Tiwari R, Prasanna R, Saxena AK (2014) Evaluating the Diversity of Culturable Thermotolerant Bacteria from
Four Hot Springs of India. J Biodivers Biopros Dev 1: 127. doi:10.4172/ijbbd.1000127
Page 2 of 9
In the present investigation on diversity of thermotolerant bacteria
in four hot springs of India (Bakreshwar, Balrampur, Chumathang and
Vashist), a culture based method complementing with ARDRA and
16S rRNA gene sequencing was used. Niche specific taxonomic groups
of bacteria were defined. A set of selected highly thermo tolerant
strains were further characterized using Biolog.
Materials and Methods
Sampling and Isolation of bacteria
Water samples were collected from four hot springs and brought to
the laboratory in thermos flasks within 12 hours. These samples were
then processed for the isolation of culturable bacteria. The physicochemical characteristics of the water samples were tested in terms of
pH and temperature. Five different media were used for enumeration
and isolation, using standard spread plate technique and incubated at
37°C for 48-72 h. The composition of the different media employed
were - Nutrient Agar (Peptone 0.5%, Beef extract 0.3%, NaCl 0.5% and
Agar 1.8%), Thermus Agar (Peptone 0.5%, Yeast extract 0.2%, Beef
extract 0.4%, NaCl 0.5% and Agar 1.8%), R2A medium (Proteose
Peptone 0.05%, Casamino acid 0.05%, Yeast extract 0.05%, Dextrose
0.05%, Soluble starch 0.05%, Dipotassium hydrogen phosphate 0.03%,
Sodium Pyruvate 0.03%, Magnesium sulphate heptahydrate 0.005%),
King’s B medium (Protease Peptone 2%, Dipotassium hydrogen
phosphate 0.15%, Magnesium sulphate 0.15% and Agar 1.8%) and
Thermus Peptone Meat extract Yeast extract medium (TPMY :
Peptone 0.35%, Meat extract 0.5% Yeast extract 0.2% , NaCl 0.15% and
Agar 1.8%). The total viable count was recorded in the different media.
research [22]. Ever since Thomas Brock discovered the presence of
Thermus aquaticus in the thermal vents of Yellowstone National Park,
a number of research groups started exploring similar environments
all around the world [2,5-7,23]. Despite intensive studies on
geothermal springs, scanty information is available on diversity of
bacteria in moderate altitudes or high altitude regions.
Indian subcontinent is a host to innumerable number of geothermal
hot springs, especially the Himalayan Geothermal Belt which contains
close to 150 thermal springs. Chumathang which is at an elevation of
4023 m above mean sea level and Vashist, which is at an elevation of
1982 m come under HGB. Bakreshwar hot springs present in eastern
India, is at an elevation of 84 m above mean sea level and Balrampur
hot springs present in central India, is at an elevation of 623 m above
mean sea level. Our investigation focussed on the diversity of
culturable thermotolerant bacteria from hot springs present in both
the high altitude regions and plains. Chumathang, Vashist and
Balrampur hot springs represent high altitude regions in the
descending order of altitude and Bakreshwar represents the
geothermal springs of plains (Figure 1). Among the different samples,
the temperature was highest in the Balrampur hot springs and lowest
in Vashist hot springs.
Screening for temperature tolerance
All the isolates from each of the four springs were screened for
temperature tolerance by spotting the cultures on specific media at
different temperatures viz 40, 45, 50, 55 and 60°C for 72 h. Isolates
growing at 60°C on solid media were tested further at temperatures
beyond 60°C in liquid media by incubating them in water bath-shaker.
Molecular techniques and Bioinformatics analysis
Methods of DNA extraction, PCR amplification of 16S rRNA gene,
ARDRA, 16S rRNA gene sequencing and phylogenetic analysis were as
followed in our earlier studies [21].
Biolog analyses of selected isolates
A set of isolates capable of growing upto a temperature of 60° C
were characterized using the GEN III MicroPlate™, to generate a profile
based on 94 biochemical tests (70 carbon sources and 22 chemical
sensitivity tests). Biolog’s microbial identification system software was
used to identify the bacterium from its phenotypic pattern in the GEN
III MicroPlate.
Results and Discussion
Hot springs are manifestations of geological activity and represent
extreme environments which have been mainly explored in terms of
their physico-chemical characteristics. Among the different extreme
environments, thermal springs are analogous to primitive earth and
hydrothermal processes on Mars. Hence, the microbial communities
thriving in geothermal hot springs have been the subject of extensive
J Biodivers Biopros Dev
ISSN: IJBBD, an open access journal
Figure 1: Map showing the location and physico-chemical
characteristics of samples.
Enumeration and isolation of culturable bacteria
Five different types of media were employed to capture the
maximum possible diversity. These included a nutrient rich medium
(Nutrient Agar) suitable for most nutritional types, a medium suitable
for oligotrophs (R2A), specific media designed for thermophiles
(Thermus Agar and Thermus Peptone Meat extract Yeast extract
medium) and a medium mostly used for Pseudomonas and related
genera (King’s B medium). Enumeration of bacterial population
revealed that aerobic heterotrophic bacteria in Bakreshwar thermal
springs ranged from 119 to 183 × 104 CFU/mL and a similar trend was
Volume 1 • Issue 3 • 1000127
Citation:
Kumar M, Nath Yadav A, Tiwari R, Prasanna R, Saxena AK (2014) Evaluating the Diversity of Culturable Thermotolerant Bacteria from
Four Hot Springs of India. J Biodivers Biopros Dev 1: 127. doi:10.4172/ijbbd.1000127
Page 3 of 9
recorded for Vashist thermal springs, which was in the range of 158 to
229 × 104 CFU/mL. Among the different media used, R2A yielded
highest population of 229 × 104 CFU/mL from Vashist samples and
for Bakreshwar samples, while TPMY yielded the highest population
of 183 × 104 CFU/mL. In the samples from Balrampur and
Chumathang, the population of aerobic heterotrophs were in the range
from 89 to 193 × 103 CFU/mL and 69 to 112 × 104 CFU/mL
respectively. Thermus agar yielded highest population from both
Balrampur and Chumathang samples. A total of 60, 48, 46 and 52
morphotypes were selected from Bakreshwar, Balrampur,
Chummathang and Vashist hot spring samples respectively, for
further studies.
representative isolates of Vashist also belonged to Bacillus. In
Balrampur samples, out of 14 representative isolates, 12 belonged to
the genus Bacillus. A taxonomic grouping of the isolates revealed that
Firmicutes was the dominant division in both Bakreshwar and
Balrampur, but Chumathang and Vashist hot water springs exhibited a
greater diversity with both containing members from Firmicutes,
Proteobacteria and Actinobacteria. Actinobacterial members were
present in all the four hot springs studied and Chumathang hot
springs recorded highest number i.e. 18% of the sequenced isolates.
Members of Proteobacteria were present only in Chumathang and
Vashist hot springs and members of Bacteroidetes were found only in
Bakreshwar hot springs (Figure 2).
Screening for temperature tolerance
Screening for temperature tolerance revealed a total of 27, 38, 18,
and 29 isolates from Bakreshwar, Balrampur, Chumathang and
Vashist respectively were able to grow at 45°C. Seven isolates were able
to grow at 50°C, while five were able to grow at 55°C and three were
able to grow at 60°C. Only one isolate was able to grow at 70°C.
Interestingly, only the isolates from Bakreshwar were able to grow at
temperatures beyond 45°C, although Bakreshwar hot springs did not
exhibit the highest temperature among the four hot springs. The
microbial community obtained from these hot springs includes
mesophiles also, which reveals the possibility of beneficial partnerships
of such high temperature adapted mesophiles with more
thermotolerant non culturable bacteria, as has been recorded in several
extreme habitats [5]. This is in consonance with reports regarding the
absence of a monotonic relation between temperature stress and
microbial community, and illustrating that thermophiles can be
members of mesophilic environments also and vice versa [5,24].
Metagenomic analyses can help further throw light on our
observations. Isolates which were able to grow at 45°C and above were
selected for molecular characterization.
Amplified rDNA restriction analysis
PCR amplification of 16S rRNA gene yielded a single amplicon of
1.5 Kbp from all the isolates. The amplicons were digested with three
restriction enzymes. Different patterns comprising of 3 to 6 fragments
ranging in size from 100 to 800 bp, were characterized. RFLP analysis
revealed that restriction digestion with Alu I was more discriminative,
as compared to Msp I and Hae III. A combined dendrogram (based on
patterns generated using three enzymes individually) was constructed
to determine the percent similarity among the isolates from each of the
three thermal springs (data not shown). At 90% similarity level,
thermotolerant isolates from Bakreshwar, Balrampur, Chumathang
and Vashist thermal springs grouped into 14, 14, 11 and 9 clusters
respectively. ARDRA has been effectively utilized in the past for both
culture dependent and culture independent diversity studies as a tool
to group the isolates and clones into phylotypes [17-19].
16S rRNA gene sequencing and phylogenetic analysis
Representative isolates from each phylogenetic cluster were
sequenced and the data were analysed by BLAST and the nearest
match from the GenBank database is illustrated (Table 1). Among the
14 isolates sequenced from Bakreshwar hot springs, 10 belonged to the
genera Bacillus. Sequence results from Chumathang hot springs
revealed that 4 out of 11 isolates were Bacillus. Two isolates of the 9
J Biodivers Biopros Dev
ISSN: IJBBD, an open access journal
Figure 2: a Distribution of different Divisions of Eubacteria in
Bakreshwar Thermal springs; b Distribution of different Divisions
of Eubacteria in Balrampur Thermal springs; c Distribution of
different Divisions of Eubacteria in Chumathang Thermal springs;
d Distribution of different Divisions of Eubacteria in Vashist
Thermal springs.
Phylogenetic trees were constructed from the sequences of the
representative isolates from each of the four thermal springs along
with closest sequences in the NCBI Genebank (Figure 3-6). The use of
16S rRNA gene sequencing in combination with ARDRA for grouping
into phylogenetic clusters and identification of bacteria is well
documented [18-20]. Although the number of phylogenetic clusters is
less in Vashist, the average bacterial population was highest in Vashist
and the lowest in Balrampur.
This may be related to the variation in the temperature of the
springs, which was least for Vashist and highest for Balrampur.
Temperature showed a significant negative correlation with CFU
(r=-0.9950). It is interesting to note that the pH of Vashist hot spring
is also in the neutral range, suggesting the role of temperature and pH
in bacterial abundance in geothermal waters, which is in accordance
with earlier investigations [25].
Volume 1 • Issue 3 • 1000127
Citation:
Kumar M, Nath Yadav A, Tiwari R, Prasanna R, Saxena AK (2014) Evaluating the Diversity of Culturable Thermotolerant Bacteria from
Four Hot Springs of India. J Biodivers Biopros Dev 1: 127. doi:10.4172/ijbbd.1000127
Page 4 of 9
Figure 3: Unrooted phylogenetic tree based on comparison of 16S
rDNA sequences of 14 isolates of Bakreshwar along with their
closest phylogenetic relatives. Phylogenetic tree was constructed
based on aligned datasets using Neighbour joining (NJ) method
using the program MEGA 4.0.2. Numbers on the tree indicates
percentage of bootstrap sampling derived from 1000 random
samples.
Figure 4: Unrooted phylogenetic tree based on comparison of 16S
rDNA sequences of 14 isolates of Balrampur along with their
closest phylogenetic relatives. Phylogenetic tree was constructed
based on aligned datasets using Neighbour joining (NJ) method
using the program MEGA 4.0.2. Numbers on the tree indicates
percentage of bootstrap sampling derived from 1000 random
samples.
J Biodivers Biopros Dev
ISSN: IJBBD, an open access journal
Figure 5: Unrooted phylogenetic tree based on comparison of 16S
rDNA sequences of 11 isolates of Chumathang along with their
closest phylogenetic relatives. Phylogenetic tree was constructed
based on aligned datasets using Neighbour joining (NJ) method
using the program MEGA 4.0.2. Numbers on the tree indicates
percentage of bootstrap sampling derived from 1000 random
samples.
Figure 6: Unrooted phylogenetic tree based on comparison of 16S
rDNA sequences of 9 isolates of Vashist along with their closest
phylogenetic relatives. Phylogenetic tree was constructed based on
aligned datasets using Neighbour joining (NJ) method using the
program MEGA 4.0.2. Numbers on the tree indicates percentage of
bootstrap sampling derived from 1000 random samples.
Volume 1 • Issue 3 • 1000127
Citation:
Kumar M, Nath Yadav A, Tiwari R, Prasanna R, Saxena AK (2014) Evaluating the Diversity of Culturable Thermotolerant Bacteria from
Four Hot Springs of India. J Biodivers Biopros Dev 1: 127. doi:10.4172/ijbbd.1000127
Page 5 of 9
Representative
isolates
Temperature tolerance
GeneBank
number
1
B1
50°C
KC492101
Bacillus cereus (GQ375259)
99
Firmicutes
2
B3
60°C
KC492102
Bacillus pumilus (JX841107)
99
Firmicutes
3
B4
JX312592
Bacillus
(JN366726)
licheniformis
99
Firmicutes
4
B6
JX312593
Pontibacter
(FJ897494)
niistensis
98
Bacteroidetes
5
B9
JX312594
Bacillus sp. (DQ448753)
99
Firmicutes
6
B10
JX312595
Lysinibacillus
fusiformis(EU430993)
99
Firmicutes
7
B16
45°C
JX312596
Bacillus flexus (DQ837543)
99
Firmicutes
8
B19
45°C
KC492103
Bacillus sp. (JX402419)
99
Firmicutes
9
B21
JX312597
Bacillus
(EU931553)
99
Firmicutes
10
B25
JX312598
Exiguobacterium
(AY745848)
99
Firmicutes
11
B27
JX312599
Bacillus sp.(EU584536)
99
Firmicutes
12
B31
KC492104
Bacillus
(AB301007)
99
Firmicutes
13
B36
45°C
KC492106
Bacillus sp. (AB681430)
99
Firmicutes
14
B38
70°C
KC492107
Kocuria sp. (JF834545)
100
Actinobacteria
1
BC1
JX312575
45°C
Bacillus cereus (JQ739719)
100
Firmicutes
2
BC2
JX312576
45°C
Brevibacillus sp. (AJ313027)
99
Firmicutes
3
BC5
JX312578
45°C
Bacillus pumilus (JQ435673)
99
Firmicutes
4
BC6
JX312579
45°C
Bacillus
(HQ242769)
100
Firmicutes
5
BC7
JX312580
45°C
Bacillus firmus (JQ249909)
100
Firmicutes
6
BC9
JX312581
45°C
Bacillus
(EU931553)
100
Firmicutes
7
BC10
JX312582
45°C
Bacillus sp. (HM567109)
100
Firmicutes
8
BC11
JX312583
45°C
Bacillus flexus (HQ875778)
99
Firmicutes
9
BC12
JX312584
45°C
Planococcus sp. (EF471920)
100
Firmicutes
10
BC16
JX312587
45°C
Bacillus
(GU377281)
99
Firmicutes
11
BC17
JX312588
45°C
Lysinibacillus sp. (FN397524)
100
Firmicutes
RFLP Pattern
Representative
isolates
Temperature tolerance
GeneBank accession
Nearest
neighbour
12
BC21
RFLP Pattern
accession Nearest
neighbour
phylogenetic 16 Similitude
Division
(%)
Bakreshwar
45°C
45°C
45°C
45°C
50°C
55°C
55°C
60°C
megaterium
sp.
licheniformis
Balrampur
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ISSN: IJBBD, an open access journal
number
JX312589
45°C
Staphylococcus
(JQ624771)
aryabhattai
megaterium
licheniformis
phylogenetic 16 Similitude
Division
(%)
haemolyticus
99
Firmicutes
Volume 1 • Issue 3 • 1000127
Citation:
Kumar M, Nath Yadav A, Tiwari R, Prasanna R, Saxena AK (2014) Evaluating the Diversity of Culturable Thermotolerant Bacteria from
Four Hot Springs of India. J Biodivers Biopros Dev 1: 127. doi:10.4172/ijbbd.1000127
Page 6 of 9
13
BC27
14
BC31
JX312590
45°C
Rhodococcus sp.( DQ285075)
99
Actinobacteria
JX312591
45°C
Exiguobacterium
(HM047519)
100
Firmicutes
45°C
JX312600
Paracoccus sp. (AY864654)
99
Proteobacteria
JX312601
Cellulosimicrobium
(EU931556)
99
Actinobacteria
JX312602
Aurantimonas
(AB682666)
99
Proteobacteria
acetylicum
Chumathang
1
LC4
2
LC5
3
LC7
4
LC8
45°C
JX312603
Bacillus pumilus (JF803782)
99
Firmicutes
5
LC10
45°C
JX312604
Kocuria palustris (HM218471)
100
Actinobacteria
6
LC11
45°C
JX312605
Klebsiella sp. (GU290319)
100
Proteobacteria
7
LC13
45°C
JX312607
Bacillus sp. (JF901711)
100
Firmicutes
8
LC15
KC492091
Bacillus
(JX997394 )
99
Firmicutes
9
LC17
JX312609
Brevundimonas
(NR_043726)
terrae
100
Proteobacteria
10
LC19
JX312610
Staphylococcus
(JN315890)
arlettae
100
Firmicutes
11
LC24
45°C
JX312612
Bacillus firmus (JQ249909)
100
Firmicutes
1
V1
45°C
KC492092
Bacillus pumilus (HF536558)
100
Firmicutes
2
V6
KC492093
Acinetobacter
(JQ337945)
99
Proteobacteria
3
V7
KC492094
Acinetobacter junii (JX307680)
100
Proteobacteria
4
V8
KC492095
Enterobacter
(JF894166)
100
Proteobacteria
5
V13
KC492096
Kocuria sp. (JF778721)
99
Actinobacteria
6
V21
KC492097
Exiguobacterium
(GU397441)
99
Firmicutes
7
V26
KC492098
Bacillus subtilis (JX502843)
99
Firmicutes
8
V28
KC492099
Gulbenkiania
(NR_042548)
99
Proteobacteria
9
V29
KC492100
Brevibacillus sp. (DQ116777)
100
Firmicutes
45°C
45°C
45°C
45°C
45°C
cellulans
altamirensis
megaterium
Vashist
45°C
45°C
45°C
45°C
45°C
45°C
45°C
45°C
baumanii
cloacae
sp.
mobilis
Table 1: RFLP patterns and sequenced representative isolates of thermal springs.
In the present study, 16S rRNA gene sequencing of representative
isolates from each phylotype, followed by BLAST search revealed that
a majority of sequences from both Balrampur and Bakreshwar (71.4%
and 85.7% respectively) showed closeness to Bacillus, whereas only
36.4% and 22.2% of the sequenced isolates from Chumathang and
Vashist respectively showed closeness to Bacillus. Apart from Bacillus,
representatives from Bacillus derived genera were present in both
Bakreshwar (Brevibacillus and Lysinibacillus) and Vashist
(Brevibacillus) hot springs. The members of the genus Bacillus are
spore formers and also produce a number of biocidal metabolites/
J Biodivers Biopros Dev
ISSN: IJBBD, an open access journal
enzymes which makes them a common inhabitant of diverse extreme
habitats [26]. Sufficient evidence exists for reclassification of
thermophilic bacteria in the genus Bacillus into Brevibacillus,
Aneurinibacillus, Amphibacillus, Virgibacillus, Alicyclobacillus,
Paenibacillus, Halobacillus and Geobacillus based on the 16S rRNA
gene sequence [15].
A majority of sequenced isolates from all the four hot springs
belonged to Firmicutes division. It was found that 86%, 93%, 44% and
44% of the sequenced isolates respectively from Bakreshwar,
Balrampur, Chumathang and Vashist belonged to Firmicutes (Figure
Volume 1 • Issue 3 • 1000127
Citation:
Kumar M, Nath Yadav A, Tiwari R, Prasanna R, Saxena AK (2014) Evaluating the Diversity of Culturable Thermotolerant Bacteria from
Four Hot Springs of India. J Biodivers Biopros Dev 1: 127. doi:10.4172/ijbbd.1000127
Page 7 of 9
2). The bacterial communities in many hot springs are dominated by
Firmicutes [10,11,22]. Another commonality in these four hot springs
is the presence of isolates belonging to Actinobacteria. Gram positive
prokaryotes are known to be comparatively stress resistant and long
range migrants, especially the Firmicutes and Actinobacteria [27].
It is interesting to note that bacteria belonging to the division
Proteobacteria were isolated from the hot springs in Himalayan
Geothermal Belt (Chumathang and Vashist) alone. Bacterial
communities in the hot springs of high elevated regions of Tibet are
known to be predominated by Proteobacteria, alongside Firmicutes
[10] which supports our observation. Bacteroidetes is present only in
the Bakreshwar which is a representative of plain. The genus in this
division is Pontibacter which is normally a soil bacterium [28] and has
not been isolated from hot springs. The presence of Pontibacter
suggests a migration of bacterial communities from the sediments to
the spring water aided by the flow of springs through the sediments. It
is interesting to note that the three hot springs belonging to high
altitude region exhibit high level of diversity of bacteria. Chumathang
exhibited the highest number of different genera, while Bakreshwar
had the least number. This observation supplemented with the
presence of Proteobacteria in the hot springs of Himalayan
Geothermal Belt, reemphasizes the effect of geographic location on the
type of bacterial communities in any particular environment [29].
The isolates from Chumathang hot springs showed closeness to
many bacteria like Bacillus and its derived genera and genera like
Paracoccus, Kocuria, Klebsiella and Staphylococcus. These genera
were also recorded in our earlier study on diversity of thermotolerant
bacteria in Manikaran hot springs [21] which also falls under HGB.
Cellulosimicrobium,
Besides
these,
representatives
from
Aurantimonas and Brevundimonas were niche specific to
Chumathang hot springs. Cellulosimicrobium is very closely related to
Cellulomonas. Schuman et al. [30] suggested that Cellulomonas
should be reclassified as Cellulosimicrobium. Cellulomonas has
previously been reported as a culturable thermoresistant aquatic
bacterium from the oligotrophic pools in Mexico [27]. To our
knowledge, this is the first report on Aurantimonas and
Brevundimonas in a hot spring.
earlier study on diversity of thermotolerant bacteria from Manikaran
[21] and also in the studies on the diversity of culturable
thermoresistant aquatic bacteria in Mexico [27]. Representatives from
Acinetobacter, Enterobacter and Gulbenkiania, which are pathogens
and associated with human intestine, may have been transmitted as
contaminants from people taking shower in the spring, as this spring is
known for its medicinal properties. The genera Exiguobacterium,
Staphylococcus and Brevibacillus were also present in Balrampur hot
springs. There are also representations from Planococcus and
Rhodococcus, both being niche specific to Balrampur, but have also
been reported in our early study on Manikaran hot springs [21].
Bakreshwar, which showed least diversity with reference to number of
genera obtained, has representatives from Exiguobacterium, Kocuria,
Lysinibacillus and Pontibacter; among which Pontibacter was niche
specific to Bakreshwar.
Biochemical tests and substrate utilization profiles for
selected isolates using Biolog
The data on substrate utilization patterns and sensitivity to
chemicals for the 3 temperature tolerant isolates from Bakreshwar
were tabulated. Significant variations were observed among the
isolates in terms of utilization of sugars, sugar derivatives, metabolic
intermediates and amino acids and peptides. Among the three isolates
tested for a total of 20 sugars, isolate B31 was able to utilize all, except
one sugar. α-D-Glucose, sucrose and pectin were utilized by all the
three isolates. Generally known as refractile substrates and not
commonly utilized by bacteria, compounds such as D-turanose, Dglucuronic acid, glucuronamide, mucic acid, quinic acid, D-saccharic
acid, formic acid and D-serine were not utilized by any of the three
isolates. Isolate B31 was able to utilize maximum number of sugar
derivatives [15], followed by B3 which was able to utilize 9 of the 19
sugar derivatives tested (Table 2). Out of 19 metabolic intermediates
tested, 15 were found utilized by B38 and B31 utilized 13 sugar
derivatives (Table 2). The results on amino acids and peptides
utilization revealed that the isolate B31 was able to utilize a maximum
of 11 amino acids followed by B3, which utilized 7 amino acids (Table
2).
Among the different genera obtained from Vashist hot springs
Exiguobacterium, Kocuria and Brevibacillus were reported in our
Resistance to antimicrobials and growth
conditions
Substrates Utilized
Isolates
(19)
Amino
acids
Antimicrobial
and
peptides
compounds (17)
(12)
Growth conditions
(5)
9
8
7
5
5
19
15
13
11
4
5
9
8
15
2
9
4
Saccharides
Metabolic intermediates
(20)*
Saccharide
derivatives (19)
B3
9
B31
B38
Table 2: Profiling of the promising isolates using BIOLOG.
Among the three isolates tested for 17 antimicrobial compounds,
the isolate B38 was found to be resistant to nine antimicrobial
compounds. All the three isolates exhibited resistance to aztreonam
and potassium tellurite. The results also revealed that the isolates were
sensitive to five antimicrobial compounds. All the three isolates were
able to show growth at pH up to 6 and salt concentration up to 8%.
J Biodivers Biopros Dev
ISSN: IJBBD, an open access journal
B38 is the lone isolate which was not able to show growth at pH 5.
Biolog based phenotyping of the three highly thermotolerant isolates
generated useful unique fingerprints of the isolates (Table 3), besides
providing valuable information on the utilization of routine and rare
substrates and sensitivity/resistance to antimicrobial compounds.
Volume 1 • Issue 3 • 1000127
Citation:
Kumar M, Nath Yadav A, Tiwari R, Prasanna R, Saxena AK (2014) Evaluating the Diversity of Culturable Thermotolerant Bacteria from
Four Hot Springs of India. J Biodivers Biopros Dev 1: 127. doi:10.4172/ijbbd.1000127
Page 8 of 9
Among the three isolates, two belonged to the genus Bacillus and one
belonged to the genus Turicella.
Antimicrobial Compounds
Isolates
Substrates
Sensitive to
Resistant to
Lithium chloride
Guanidine HCl and Sodium
Butyrate
B3
D-Arabitol,
B31
L-Fucose, L-Rhamnose, D-Melibiose, Stachyose, N-Acetyl-β-D-Mannosamine, NAcetyl-D-Galactosamine, N-Acetyl Neuraminic acid, L-Histidine, L-Pyroglutamic acid, Inosine and Glycyl-L-Proline
-
B38
β-Hydroxy - Butyric acid
Minocycline, Fusidic acid and
Sodium Bromate
Sodim lactate
Table 3: Unique traits of promising isolates in terms of substrate utilization and response to antimicrobial compounds.
Biolog based identification of temperature tolerant isolates matched
with 16S rDNA sequence based identification up to genera level only
for two isolates. One isolate for which there was no match was B38,
which according to Biolog analysis identifies it as Turicella otitidis, but
according to 16S rRNA gene sequence, it was placed in the genus
Kocuria. Morphotyping of isolate B38 revealed its ability to utilize ßhydroxy butyric acid and unique resistance profile against
minocycline, fusidic acid and sodium bromate. Earlier studies using
have shown its utility in differentiation of Bacillus species [31].
However Biolog analyses have certain limitations, as it is unable to
distinguish closely related organisms [32]. Also the database of
Actinobacteria in Biolog is weak hence a clear cut identification of
Isolate B38 was difficult; however this strain can have tremendous
biotechnological potential.
The identification of isolate B3 using both Biolog analysis and 16S
rRNA gene identity matched and placed it as Bacillus pumilus. B3 was
able to utilize only 35.71% of the substrates tested. Strains of Bacillus
pumilus generally show high resistance to environmental stresses,
including UV light exposure, desiccation, and the presence
of oxidizers such as hydrogen peroxide and produce compounds
antagonist to fungal and bacterial pathogens [17]. In the present
investigation, this strain was able to show growth on 45% of the
substrates and chemical agents tested, besides being uniquely able to
use D-Arabitol, and uniquely exhibiting resistance to guanidine and
sodium butyrate (Table 3).
Isolate B31, identified as Bacillus licheniformis based on Biolog, was
found to show closest sequence based identity with Bacillus subtilis.
This isolate was able to uniquely utilize 11 substrates. Rey et al. [33]
determined the complete genome sequence of B. licheniformis ATCC
14580 and compared the information generated with other species of
Bacillus, notably with B. subtilis 168. Although there exists
unmistakable organizational similarities (84.6% identical at the
nucleotide level) between the B. licheniformis and B. subtilis genomes,
notable differences in the numbers and locations of prophages,
transposable elements and a number of extracellular enzymes and
secondary metabolic pathway operons have aided in distinguishing
these species. The isolate B31 corresponding to Bacillus licheniformis
was able to utilize 82.85% of the substrates tested while isolate. Indepth analyses of our isolate may provide more definitive information.
Our study clearly illustrated the significance of evaluating microbial
diversity using a combination of traditional morphotyping, followed
by molecular approaches and BIOLOG analyses of promising isolates.
J Biodivers Biopros Dev
ISSN: IJBBD, an open access journal
Niche specific taxa or genera were also identified. These promising
isolates can be of immense significance, as their unique substrate
utilization and antibiotic resistance profiles, can make them
competitive in the environment.
Acknowledgements
The first author is thankful to Department of Science and
Technology, Government of India for providing fellowship for his
Ph.D program and Post Graduate School, IARI. All the authors are
thankful to the Division of Microbiology, IARI, New Delhi for
providing necessary facilities for undertaking this study. The financial
support from National Agricultural Innovation Project (Indian
Council of Agricultural Research) under the project “Diversity analysis
of Bacillus and other predominant genera in extreme environments
and its utilization in Agriculture” is gratefully acknowledged. The
authors also sincerely thank the technical assistance of Central
Instrumentation Facility, Delhi University for BIOLOG analysis.
References
1.
2.
3.
4.
5.
6.
7.
8.
Satyanarayana T, Raghukumar C, Shivaji S (2005) Extremophilic
microbes: Diversity and perspectives. Curr Sci 89: 78-90.
Sayeh R, Birrien JL, Alain K, Barbier G, Hamdi M, et al. (2010) Microbial
diversity in Tunisian geothermal springs as detected by molecular and
culture-based approaches. Extremophiles 14: 501-514.
Ghosh D, Bal B, Kashyap VK, Pal S (2003) Molecular phylogenetic
exploration of bacterial diversity in a Bakreshwar (India) hot spring and
culture of Shewanella-related thermophiles. Appl Environ Microbiol 69:
4332-4336.
Jha M (1992) Hydrobiological Studies on Suraj Kund and Chandrama
Kund, Hot Springs of Rajgir, Bihar, India. Int Rev Hydrobiol 77: 435-443.
Mitchell KR (2009) Controls on microbial community structure in
thermal environments: exploring bacterial diversity and the relative
influence of geochemistry and geography. Dissertation, University of
New Mexico.
Reigstad LJ, Jorgensen SL, Schleper C (2010) Diversity and abundance of
Korarchaeota in terrestrial hot springs of Iceland and Kamchatka. ISME J
4: 346-356.
Aditiawati P, Yohandini H, Madayanti F, Akhmaloka (2009) Microbial
diversity of acidic hot spring (kawah hujan B) in geothermal field of
kamojang area, West Java-Indonesia. Open Microbiol J 3: 58-66.
Pathania S, Sharma N, Verma SK (2012) Optimization of cellulase-free
xylanase produced by a potential thermoalkalophilic Paenibacillus sp. N1
isolated from hot springs of northern himalayas in India. J Microbiol
Biotechnol Food Sci 2: 1-24.
Volume 1 • Issue 3 • 1000127
Citation:
Kumar M, Nath Yadav A, Tiwari R, Prasanna R, Saxena AK (2014) Evaluating the Diversity of Culturable Thermotolerant Bacteria from
Four Hot Springs of India. J Biodivers Biopros Dev 1: 127. doi:10.4172/ijbbd.1000127
Page 9 of 9
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Mukhopadhyay DK, Sarolkar PB (2012) Geochemical appraisal of
Bakreshwar-Tantloi hot springs, West Bengal and Jharkhand, India. In:
Proceedings of the Thirty-Seventh Workshop on Geothermal Reservoir
Engineering: 1-5.
Huang Q, Dong CZ, Dong RM, Jiang H, Wang S, et al. (2011) Archaeal
and bacterial diversity in hot springs on the Tibetan Plateau, China.
Extremophiles 15: 549-563.
Pagaling E, Grant WD, Cowan DA, Jones BE, Ma Y, et al. (2012)
Bacterial and archaeal diversity in two hot spring microbial mats from
the geothermal region of Tengchong, China. Extremophiles 16: 607-618.
Piterina AV, Pembroke JT (2010) Preparation and analysis of
environmental DNA: optimisation of techniques for phylogenetic
analysis of ATAD sludge. In: Mendez-Vilaz A (ed) Current Research,
Technology and Education Topics in Applied Microbiology and
Microbial Biotechnology, Formatex: 1533-1538.
Acharya S, Chaudhary A (2012) Alkaline cellulase produced by a newly
isolated thermophilic Aneurinibacillus thermoaerophilus WBS2 from hot
spring, India. Afr J Microbiol Res 6: 5453-5458.
Akanbi TO, Kamaruzaman AL, Abu-Bakar F, Hamid SAN, Radu S, Aet
al. (2010) Highly thermostable extracellular lipase-producing Bacillus
strain isolated from a Malaysian hot spring and identified using 16S
rRNA gene sequencing. Int Food Res J 17: 45-53.
Adiguzel A, Ozkan H, Baris O, Inan K, Gulluce M, et al. (2009)
Identification and characterization of thermophilic bacteria isolated from
hot springs in Turkey. J Microbiol Methods 79: 321-328.
Patel JB (2001) 16S rRNA gene sequencing for bacterial pathogen
identification in the clinical laboratory. Mol Diagn 6: 313-321.
Yadav S, Kaushik R, Saxena AK, Arora DK (2011) Diversity and
phylogeny of plant growth-promoting bacilli from moderately acidic soil.
J Basic Microbiol 51: 98-106.
Divya B, Soumya KV, Nair S (2010) 16SrRNA and enzymatic diversity of
culturable bacteria from the sediments of oxygen minimum zone in the
Arabian Sea. Antonie Van Leeuwenhoek 98: 9-18.
Sklarz MY, Angel R, Gillor O, Soares MI (2009) Evaluating amplified
rDNA restriction analysis assay for identification of bacterial
communities. Antonie Van Leeuwenhoek 96: 659-664.
Krízová J, Spanová A, Rittich B (2006) Evaluation of amplified ribosomal
DNA restriction analysis (ARDRA) and species-specific PCR for
identification of Bifidobacterium species. Syst Appl Microbiol 29: 36-44.
Kumar M, Yadav AN, Tiwari R, Prasanna R, Saxena A.K (2013)
Deciphering the diversity of culturable thermotolerant bacteria from
Manikaran hot springs. Ann Microbiol 64: 741-751.
J Biodivers Biopros Dev
ISSN: IJBBD, an open access journal
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
Tobler DJ, Benning LG (2011) Bacterial diversity in five Icelandic
geothermal waters: temperature and sinter growth rate effects.
Extremophiles 15: 473-485.
Kumar B1, Trivedi P, Mishra AK, Pandey A, Palni LM (2004) Microbial
diversity of soil from two hot springs in Uttaranchal Himalaya. Microbiol
Res 159: 141-146.
Lau MCY, Jing H, Aitchison JC, Pointing SB (2006) Highly diverse
community structure in a remote central Tibetan geothermal spring does
not display monotonic variation to thermal stress. FEMS Microbiol Ecol
57: 80–91.
Purcell D, Sompong U, Yim LC, Barraclough TG, Peerapornpisal Y, et al.
(2007) The effects of temperature, pH and sulphide on the community
structure of hyperthermophilic streamers in hot springs of northern
Thailand. FEMS Microbiol Ecol 60: 456-466.
Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P (2000)
Resistance of Bacillus endospores to extreme terrestrial and
extraterrestrial environments. Microbiol Mol Biol Rev 64: 548-572.
Cerritos R, Eguiarte LE, Avitia M, Siefert J, Travisano M (2011) Diversity
of culturable thermo-resistant aquatic bacteria along an environmental
gradient in Cuatro Cienegas, Coahuila, Mexico. A van Leeuw 99:
303-318.
Dastager SG, Raziuddin QS, Deepa CK, Li WJ, Pandey A (2010)
Pontibacter niistensis sp. nov., isolated from forest soil. Int J Syst Evol
Microbiol 60: 2867-2870.
Whitaker RJ, Grogan DW, Taylor JW (2003) Geographic barriers isolate
endemic populations of hyperthermophilic archaea. Science 301:
976-978.
Schumann P, Weiss N, Stackebrandt E (2001) Reclassification of
Cellulomonas cellulans (Stackebrandt and Keddie 1986) as
Cellulosimicrobium cellulans gen. nov., comb. nov. Int J Syst Evol
Microbiol 51: 1007-1010.
Chun S, Sneichder RW, Chung I (2003) Determination of Carbon Source
Utilization of Bacillus and Pythium Species by Biolog® Microplate Assay.
J Microbiol 41: 252-258.
Singh AK, Tripathi BM, Sahay H, Singh RN, Kaushik R, et al. (2010)
Biochemical and molecular characterization of thermo-alkali tolerant
xylanase producing bacteria from thermal springs of Manikaran. Indian J
Microbiol 50: S2-S9.
Rey MW, Ramaiya P, Nelson BA, Brody-Karpin SD, Zaretsky EJ, et al.
(2004) Complete genome sequence of the industrial bacterium Bacillus
licheniformis and comparisons with closely related Bacillus species.
Genome Biol 5: R77.
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